Methods for separation and detection of ketosteroids and other carbonyl-containing compounds

ABSTRACT

Methods for enhancing detection by mass spectroscopy (MS) and/or chromatographic separability of carbonyl-containing compounds such as steroids are disclosed. Reaction of a carbonyl compound with a sulfonhydrazide compound provides a sulfonhydrazone with enhanced ionization efficiency during the electrospray ionization process. In a particularly disclosed embodiment, derivatization of catechol estrogens with p-toluenesulfonhydrazide enhances both detection by atmospheric pressure ionization-MS (API-MS), such as electron spray ionization-MS (ESI-MS) and separation by liquid chromatography (such as HPLC) under reverse phase conditions. In yet other embodiments, the sulfonhydrazone is further reacted with a sulfonyl halide under alkaline conditions to derivatize hydroxyl groups in the compound. Prior formation of the sulfonhydrazide derivative protects the carbonyl bond of the compound during subsequent alkaline reaction with the sulfonyl halide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US03/11562, filed Apr. 15, 2003, which was published under PCTArticle 21(2), which in turn claims the benefit of U.S. ProvisionalApplication No. 60/372,848 filed Apr. 15, 2002. Both of these priorapplications are incorporated by reference herein.

FIELD

Methods of separating and detecting carbonyl-containing compounds,including ketosteroids, are disclosed.

BACKGROUND

Non-immunochemical qualitative and quantitative determinations ofcompounds found in biological samples often require separation of thecompound of interest from others in the sample. Once separated, thecompound of interest may be detected and/or identified by measuring someproperty of the compound.

Since many biological molecules have low volatilities and decompose whenheated, rather than vaporizing to form gas phase molecules, separationby gas chromatography (GC) is often impossible without first preparingvolatile derivatives. Therefore, high performance liquid chromatography(HPLC) is often chosen for separations of non-volatile biologicalmolecules. In HPLC, separations are made in the liquid phase andderivatization is typically unnecessary because most molecules aresoluble in at least one solvent.

Nonetheless, derivatization is useful in HPLC to enhance separation ofmolecules and to increase sensitivity of detection of the separatedmolecules. For example, the analyte molecules might not possess physicalproperties that can be accurately measured in the presence of solventmolecules. Detection of the analyte may be improved by derivatizationwith reagents to form readily detectable derivatives. For example, ananalyte can be derivatized with a fluorescent compound to make itreadily detectable.

Coupling a chromatographic method of separation with mass spectrometricdetection permits separation of complex mixtures into their components,detection of the components, and identification of the components fromtheir mass spectra. Since mass spectrometry (MS) requires conversion ofanalyte molecules to gas phase ions, coupling an HPLC column to a massspectrometer requires a means of isolating the analyte molecules fromexcess liquid solvent as they emerge from the column. If the solventwere introduced into the vacuum of a mass spectrometer, the pressureincrease would prevent the instrument from functioning. MS also requiresthat isolated analyte molecules be ionized before they can be detected.Electrospray ionization (ESI) is one method for coupling the effluent ofan HPLC column to a mass spectrometer. ESI functions to remove solventfrom a liquid sample without losing the analytes and to ionize theanalytes.

In ESI, a stream of analyte-containing solvent is passed through anarrow capillary tube, the end of which is held at a high positive ornegative electrical potential. The strong electric field that surroundsthe end of the capillary tube causes the emerging liquid to leave thecapillary as a fine mist of droplets. The droplets acquire an excess ofcharge (positive or negative depending upon the potential applied to thecapillary) as they leave the capillary and enter an atmospheric pressureevaporation chamber (ESI is an example of an atmospheric pressureionization, or API, method). As solvent continues to evaporate from thedroplet the charge density in each droplet continues to increase.Eventually, repulsion between ions in the droplet exceeds the surfacetension of the droplet and ions are expelled into the gas phase in aprocess termed ion evaporation. Before the analyte ions formed in theevaporation chamber are selectively introduced into the massspectrometer, they collide with other ions and neutral molecules. Duringthese collisions, charges may be transferred between species to form newions and new neutral molecules in a process called chemical ionization.

In positive ion mode ESI (i.e. the capillary is held at a high positivepotential), an important chemical ionization process is transfer ofprotons (H⁺) between species in the evaporation chamber. Positive ionmode ESI is widely used for vaporizing and ionizing biological moleculesbecause biological molecules typically have multiple sites in theirstructures that have an affinity for protons. Proteins, in fact, canattract and hold enough protons and other cations (e.g. Na⁺) during theESI process that they can form multiply charged ions. Smaller biologicalmolecules, however, may not ionize efficiently, especially if they donot possess groups of atoms having an affinity for protons.Derivatization of such molecules offers one way to improve theefficiency of ionization in ESI and hence detection by MS.

In combined HPLC-ESI-MS, both separability in the HPLC column anddetection by ESI-MS determine whether the method may be used todetermine particular analytes. Derivatization to improve separation byHPLC can have a detrimental effect on detection by ESI-MS, and theconverse is true, making it difficult to find appropriate derivatizationschemes for HPLC-EIS-MS.

SUMMARY

Sensitive methods for measuring ketosteroids in biological samples aredescribed. Derivatization of ketosteroids with a sulfonhydrazidecompound improves both separation and detection of ketosteroids by massspectrometry, such as ionization spectroscopy, for example API-MS suchas HPLC-API-MS, and more particularly HPLC-ESI-MS. In a specificdisclosed embodiment, derivatization with p-toluenesulfonhydrazide toform a p-toluenesulfonhydrazone compound is demonstrated to simplifyseparation and enhance detection of estrogens.

Sulfonhydrazide derivatization is also disclosed to improve ionizationof carbonyl-bearing compounds under electrospray ionization conditions.Increased ionization efficiency improves detection in a massspectrometer. For example, derivatization of catechol estrogens withp-toluenesulfonhydrazide makes it possible to quantify as little as 1nanogram catechol estrogen in a 10 mL urine sample. At this level ofquantitation, it is possible measure the low endogenous levels ofcatechol estrogens in urine from post-menopausal women.

In yet other embodiments, a second step derivatization is performed byderivatizing the ketosteroids under alkali conditions with a sulfonylcompound, such as a sulfonyl halide, for example sulfonyl chloride. Theprior reaction with the sulfonhydrazide protects the carbonyl groupagainst alkali conditions that can destroy the carbonyl group of theketosteroids in the absence of such protection. Reaction with thesulfonyl compound provides a highly ionizable moiety that enhances theirelectron spray ionization efficiency and HPLC-ESI-MS sensitivity.

In yet another embodiment, the carbonyl groups of a ketosteroid arereacted with a carbonyl protecting reagent, and the hydroxyl groups arethen reacted with a hydroxyl protecting reagent under alkali conditions.Protecting the carbonyl group of the steroids allows for thederivatization of the hydroxyl group without significant degradation,particularly alkali degradation. This two-step process of carbonyl andhydroxyl derivatizations provides better liquid chromatographyseparation of steroids, and allows for better signal detection in massspectrometry, such as API-MS (for example ESI-MS), when at least one ofthe derivatization groups contains a highly ionizable moiety thatenables ionization under either positive ion or negative ion modes ofelectrospray ionization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are HPLC ESI-MS chromatographic profiles for CE sampleswithout (A) and with (B) sulfonhydrazide derivatization.

FIGS. 2A-D are ESI mass spectra of CE-TSH (A, B) and d-CE-TSH (C,D).

FIGS. 3A-D are APCI mass spectra for CE (A, B) and d-CE (C, D) withoutderivatization.

FIGS. 4A-B are HPLC chromatographic profiles (detected using single ionmonitoring, SIM, at the indicated masses) of CE and d-CE without (A) andwith (B) TSH derivatization.

FIGS. 5A-B are HPLC-ESI-MS SIM chromatographic profiles of CE-TSH andd-CE-TSH for a 1-ng working standard (A), and a blank postmenopausalurine sample (B).

FIGS. 6A-B are graphs showing standard curves for determination of CE inurine.

FIG. 7 is a bar graph showing the urinary endogenous CE excretion inpost- and pre-menopausal women as determined by the disclosed methods.

FIG. 8 shows ESI mass spectra for the dansyl derivatives of2-hydroxyestrone (FIG. 8A), 2-hydroxyestrone-TSH (FIG. 8B);4-hydroxyestrone (FIG. 8C); and 4-hydroxyestrone-TSH (FIG. 8D).

FIG. 9 shows ESI mass spectra for the dansyl derivatives of2-hydroxyestrone-d4 (FIG. 9A); 4-hydroxyestrone-d4 (FIG. 9B);2-hydroxyestradiol (FIG. 9C); 4-hydroxyestradiol (FIG. 9D);2-hydroxylestradiol (FIG. 9E); and 4-hydroxylestradiol (FIG. 9F).

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

The following abbreviations and terms are collected here to aid thereader in understanding the description and examples which follow.

“a,” “an,” and “the” refer to one or more unless the context clearlyindicates otherwise.

Alkaline conditions: Having a pH greater than 7. In some examples highlyalkaline conditions are used, for example with a pH of greater than 9 or10.

API: Atmospheric pressure ionization. This term includes (withoutlimitation) both ESI and APCI.

HPLC—high performance liquid chromatography, a liquid chromatographicmethod of separation that includes the techniques of nano-LC andcapillary HPLC.

ESI-MS—electrospray ionization mass spectrometry, which is a particularexample of ionization spectroscopy.

LC-MS—liquid chromatography-mass spectrometry.

HPLC-ESI-MS—high performance liquid chromatography-electrosprayionization-mass spectrometry, a specific type of LC-MS in which ESI isthe ionization method.

Ionization spectroscopy: Spectroscopy preceded by ionization of theanalyte, for example by gas-phase ionization, electron ionization,chemical ionization (such as desorption chemical ionization), negativeion chemical ionization, and atmospheric pressure ionization (such aselectrospray ionization and atmospheric chemical ionization).

APCI—atmospheric pressure chemical ionization, which is another exampleof an ionization method that can be used in ionization massspectroscopy.

ketosteroid—a carbonyl-bearing steroid.

catechol estrogens (CE)—genotoxic estrogen metabolites having anaromatic ring bearing two hydroxyl groups.

dCE—deuterated analogs of catechol estrogens.

carbonyl-bearing compond—a compound having as part of its structure acarbon-oxygen double bond.

TSH—p-toluenesulfonhydrazide.

CE-TSH—catechol estrogens derivatized with p-toluenesulfonhydrazide.

dCE-TSH—deuterated catechol estrogen derivatized withp-toluenesulfonhydrazide.

detecting—a qualitative and/or quantitative measurement of a compound ina sample.

SIM—single ion monitoring.

Chromatographic separation: A separation method that depends upon thedifferent rates at which various substances moving in a stream (mobilephase) are retarded by a stationary material (stationary phase) as theypass over it. In liquid chromatography, the mobile phase is liquid.Higher performance liquid chromatography (HPLC) refers to systems whichobtain excellent resolution by forcing the mobile phase under pressurethrough a long, usually thin column. Examples of HPLC pressures are350-1500 psi, although the pressure may be higher (for example as highas 10,000 psi).

Sulfonhydrazide derivative: A compound produced by reaction with asulfonhydrazide reagent.

Sulfonyl halide derivative: A compound produced by reaction with asulfonyl halide.

A method is disclosed for detecting ketosteroids by reacting aketosteroid in a sample with a sulfonhydrazide compound to form asulfonhydrazone of the ketosteroid and analyzing the reacted sample bypositive ion mode electrospray ionization mass spectrometry. Thedetermination may be either qualitative or quantitative and is based ondetecting the sulfonhydrazone of the ketosteroid. The ketosteroid may beseparated from other components in the sample by HPLC, either before orafter derivatization, and analysis by API-MS (for example EIS-MS) isfacilitated by derivatization. Chormatography, such as liquidchromatography, for example HPLC, may be performed under reverse phase(polar solvent/non-polar stationary phase) conditions to furtherfacilitate API detection. For example, HPLC separation may beaccomplished by an isocratic or gradient elution with a methanol/watersolvent system and a C18 stationary phase. Useful gradient systemsinclude a gradient from 20:80 methanol/water to 80:20 methanol/water,for example, a gradient from 25:75 to 75:25 methanol/water, such as agradient from 40:60 to 60:40 methanol/water. In some embodiments, theketosteroid is extracted from the sample to provide a concentratedsample for analysis.

A method for enhancing the positive ion mode electrospray ionizationefficiency of a carbonyl compound is also disclosed. This methodincludes reacting a carbonyl compound with a sulfonhydrazide to form asulfonhydrazone of the carbonyl-containing compound. Sulfonhydrazonesare efficiently ionized by electrospray ionization processes making themmore easily detected by mass spectrometry. Sulfonhydrazidederivatization is particularly effective for increasing the ESI-MSsignal of ketosteroids, such as androgens, corticoids, estrogens,sterols, vitamin D metabolites, phytosteroids, neurosteroids and bileacids, and combinations thereof. In a disclosed embodiment,derivatization of catechol estrogens with p-toluenesulfonhydrazideparticularly improves their ESI-MS detection.

Examples of sulfonhydrazide compounds useful for forming detectablederivatives of carbonyl-containing compounds have the structure

wherein R is selected from the group consisting of alkyl (such as C1-C10alkyl, for example C1-C5 alkyl), substituted alkyl, aryl, andsubstituted aryl.

In some embodiments, the sulfonhydrazide may have the structure

wherein R₁-R₅ are independently selected from the group consisting ofhydrogen, C1-C5 alkyl, C1-C4 alkoxy, halogen, amino, nitro, hydroxyl,carbonyl, nitroso, cyano, and sulfonyl, and combinations thereof. Oneexample of a sulfonhydrazide having this structure isp-toluenesulfonhydrazide.

Methods for separating and detecting ketosteroids in a biologicalsample, such as a blood, urine, or tissue sample, are also provided. Aketosteroid may be extracted from a biological sample to provide aconcentrated sample of the ketosteroid, which is then reacted withp-toluenesulfonhydrazide to form a p-toluenesulfonhydrazone product ofthe ketosteroid. Separation of the p-toluenesulfonhydrazone product ofthe ketosteroid from other components in the concentrated sample isconveniently accomplished by chromatography, such as liquidchromatography, for example reverse phase HPLC. The ketosteroid may bedetected in the HPLC effluent by API-MS (such as ESI-MS).Advantageously, the p-toluenesulfonhydrazone product provides an intenseESI-MS signal that may be used to determine the presence and/or amountof the ketosteroid in the sample. In particular embodiments,quantitation of the ketosteroid is facilitated by a stable-isotopedilution method where a known amount of a deuterated analog of theketosteroid is added to the biological sample prior to extracting theketosteroid and used as an internal standard. The ketosteroid in thesample is quantified by comparing the intensity of the ESI-MS signalsfrom the ketosteroid and its deuterated analog. In a disclosedembodiment the method is applied to determine the amount of estrogen,specifically catechol estrogen, in a urine sample. Derivatization withp-toluenesulfonhydrazine also facilitates separation under convenientreverse phase HPLC condition, such as a methanol/water mobile phase anda C18 stationary phase.

For convenience, predetermined amounts of reagents and equipmentemployed to carry out the methods of the disclosure may be providedtogether in a kit in packaged combination. A kit can comprise inpackaged combination (a) a sulfonhydrazide compound and (b) otherreagents and equipment for determining the amount of ketosteroid in asample. Such other reagents and equipment include those described inExample 1 below. For example, a kit for determining the presence of aketosteroid can include a sulfonhydrazide compound and a deuteratedstandard of the ketosteroid in packaged combination. In someembodiments, the kit may also include a sulfonyl halide, such assulfonyl chloride.

The effect of derivatization with a sulfonhydrazide compound ondetectability of compounds by ESI-MS is dramatically illustrated inFIGS. 1A and 1B. FIG. 1A shows that no ESI-MS signal is observed forcatechol estrogens (CE) as they elute from the HPLC column withoutderivatization. FIG. 1B shows that with derivatization well-resolved andsymmetrical peaks corresponding to elution of the catechol estrogens areobserved.

The following examples are provided to aid understanding of thedisclosure and are not meant to limit the scope of the invention in anyway.

Example 1 HPLC-ESI-MS Determination of Catechol Estrogens

In this example, a stable isotope dilution HPLC-ESI-MS method isdescribed. The method includes a simple and rapid derivatization stepthat greatly improves method sensitivity and HPLC separability ofcatechol estrogens (CE), making LC-MS a much more competitive method forhuman endogenous catechol estrogen analysis.

A critical role for endogenous estrogen in the development of breastcancer has been postulated for more than a century, ever since Beatsondemonstrated that oophorectomy induced tumor remission in human breastcancer (Beatson, Lancet, 2: 104, 1896). Substantial evidence supports acausal relationship between risk of human breast cancer and levels ofendogenous estrogen (see, for example, Colditz, J. Natl. Cancer Inst.,90: 814, 1998). Increased risk has been reported in women with highserum and urine estrogens (see, for example, Toniolo et al., J. Natl.Cancer Inst., 87: 190, 1995), as well as in those women exposed toincreased estrogen levels over time as a result of late menopause, earlyonset of menstruation and/or postmenopausal obesity (see, for exampleHenderson et al., Cancer Res., 73: 1615, 1996). A key mechanism inestrogen-related breast cancer may be the metabolic activation ofestrogens to genotoxic metabolites called catechol estrogens (see, forexample, Yager and Liehr, Annu. Rev. Pharmacol. Toxicol., 36: 203, 1996)mainly 2-hydroxyestrone and 4-hydroxyestrone in humans. This process isshown below.

Electrophilic quinone products of these catechol estrogens can reactwith DNA to form both stable and depurinating adducts known to generatemutations and cell transformation that can initiate cancers (see, forexample, Cavalieri et al., Proc. Natl. Acad. Sci. USA, 94: 10937, 1997).It is believed that quantitative measurement of endogenous catecholestrogens will play an important role in elucidating the mechanism ofbreast carcinogenesis and in estimating the risk of breast cancer inindividual women.

Current methods for measuring endogenous catechol estrogens most ofteninvolve radioimmunoassay (RIA) (see, for example Ball et al., Steroids,33: 563, 1979; Emons et al., Acta Endocrinol., 97: 251, 1981; andMcGuinness et al., Clin. Chem., 40: 80, 1994), enzyme immunoassay (EIA)(see, for example, Klug et al., Steroids, 59: 648, 1994),high-performance liquid chromatography (HPLC) with electrochemicaldetection (see, for example, Shimada et al., J. Chromatogr., 223: 33,1981), and stable isotope dilution gas chromatography-mass spectrometry(GC-MS) (see, for example, Fotsis and Aldercreutz, J. Steroid Biochem.,28: 203, 1987).

RIA and EIA suffer from relatively poor specificity due to thecross-reactivity of antibodies (see, for example, Ziegler et al.,Environ. Health Perspect, 105(3): 607, 1997). HPLC with electrochemicaldetection has been used for catechol estrogen analysis in hamsterstreated with catechol estrogens and in pregnant women, whose estrogenlevels are elevated at least 10-fold. The stable isotope dilution GC-MSmethod is both sensitive and specific, and has been successfully usednot only for urine samples from non-pregnant premenopausal women butalso for urine from postmenopausal women, in which catechol estrogensare substantially reduced. However, the stable isotope dilution GC-MSmethod requires extensive and laborious sample preparation, includingthree C₁₈ solid phase extractions, six ion-exchange column separations,four liquid-liquid extractions, and two derivatization procedures foreach urine sample. Although liquid chromatography-mass spectrometry(LC-MS) has been used for in vitro and in vivo pharmacological studiesof catechol estrogens in rat brains (see, for example, Mitamura et al.,Analyst, 125: 811, 2000), the sensitivity of LC-MS with eitherelectrospray ionization (ESI) or atmospheric pressure chemicalionization (APCI) is not adequate for the endogenous levels of catecholestrogens in women (see, Ma and Kim, J. Am. Soc. Mass Spectrom., 8;1010, 1997).

A. Chemicals and Reagents

Catechol estrogens (CE), 2-hydroxyestrone (2-hydroxyE₁) and4-hydroxyestrone (4-hydroxyE₁), were obtained from Steraloids, Inc.(Newport, R.I., USA). Deuterium-labeled catechol estrogens (d-CE), [²H₄]2-hydroxyestrone and [²H₄] 4-hydroxyestrone, were purchased from C/D/NIsotopes, Inc. (Pointe-Claire, Quebec, Canada). The structures of eachof these compounds are shown below. All CE and d-CE analytical standardshad a purity of ≧98%.

p-Toluenesulfonhydrazide (TSH) was purchased from Aldrich Chemical Co.(Milwaukee, Wis., USA). Methanol (HPLC grade) and formic acid (reagentgrade) were obtained from EM Science (Gibbstown, N.J., USA), and water(HPLC grade) was obtained from Mallinckrodt Baker, Inc. (Paris, Ky.,USA). Glacial acetic acid (HPLC grade), L-ascorbic acid (reagent grade),boric acid (reagent grade), sodium bicarbonate (reagent grade) andsodium hydroxide (reagent grade) were purchased from J. T. Baker(Phillipsburg, N.J., USA), and sodium acetate (reagent grade) waspurchased from Fisher Scientific (Fair Lawn, N.J., USA).β-Glucuronidase/sulfatase from Helix pomatia (Type H-2) and QAE SephadexA-25 were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). Allglassware, including Pasteur pipettes, was silanized (Aqua-Sil, Pierce,Rockford, Ill., USA). QAE-Sephadex gels in acetate and borate forms wereprepared as described in Fotsis and Adlercreutz, J. Steroid Biochem.,28: 203, 1987.

B. Urine Sample Collection

Twenty-four-hour urine samples were collected in three-liter bottlescontaining 3 g ascorbic acid, to prevent oxidation, from two healthynon-pregnant premenopausal women (aged 34 and 38 years) and two healthypostmenopausal women (aged 58 and 60 years; 5+ years past last menstrualcycle). None of the women was taking exogenous estrogens. For the twopremenopausal women, samples were collected during the midfollicular(days 8-9 of menstrual cycle) and midluteal phases (6 days before theanticipated menses) of the menstrual cycle. Immediately after the urinecollection was completed, urine volume was recorded and sodium azide, toprevent bacterial growth, was added to achieve a 0.1% (w/v)concentration. One half of the 24-h urine from each of twopostmenopausal women was mixed to prepare a pooled postmenopausal urine,and the remaining two halves were non-pooled postmenopausal urines.Similarly, pooled and non-pooled premenopausal urines during eithermidfollicular or midluteal phase were prepared. Aliquots of both pooledand non-pooled urines were stored at −80° C. until analysis.

C. Preparation of Stock and Working Standard Solutions

Stock solutions of CE and d-CE were each prepared at 80 μg ml⁻¹ byaddition of 2 mg catechol estrogen powders to a volumetric flask anddiluting to 25 ml with 100% methanol. These solutions were stored at−20° C. until needed to prepare working standard solutions. During eachday of analysis, working standards of CE and d-CE were prepared byserial dilutions of stock solutions with 100% methanol. For theanalyses, d-CE working standard solution was prepared at 800 ng ml⁻¹,while CE working standard solutions were prepared at 800 and 50 ng ml⁻¹.

D. Preparation of Calibration Standards

CE are naturally present at various levels in all human urine samples,including those from males. Therefore, use of CE-spiked urine togenerate calibration curves was impractical. Non-biologic matrixcalibration standards were prepared by combining 50 μl of the d-CEworking internal standard solution (40 ng d-CE) with various volumes ofCE working standard solution, which typically ranged from 0.5 to 64 ngCE.

E. Urinary CE Hydrolysis and Extraction Procedure

To a 10-ml aliquot of urine sample, 50 μl of the d-CE working internalstandard solution (40 ng d-CE) was added, followed by 10 ml of freshlyprepared enzymatic hydrolysis buffer containing 50 mg of L-ascorbicacid, 100 μl of β-glucuronidase/sulfatase from Helix pomatia (Type H-2)and 10 ml of 0.15 M sodium acetate buffer (pH 4.1). The sample wasincubated overnight at 37° C. After hydrolysis, the sample was appliedto a primed C₁₈ column (Bond Elut LRC, Chrom Tech, Inc., Apple Valley,Minn., USA) and washed with 5 ml of water. CE and d-CE were eluted with3 ml of methanol and further purified on QAE-Sephadex in acetate andborate forms, respectively, as described by Fotsis and Adlercreutz.

F. Derivatization Procedure

The fraction containing both CE and d-CE was evaporated to dryness undernitrogen gas (Reacti-Vap III™, Pierce, Rockford, Ill., USA) andderivatized to form the CE and d-CE p-toluenesulfonhydrazones (CE-TSHand d-CE-TSH, respectively) by reaction with 400 μgp-toluenesulfonhydrazide (TSH) in 200 μl methanol and heating at 60° C.(Reacti-Therm III™ Heating Module, Pierce, Rockford, Ill., USA) for 30min. Calibration standard mixtures were derivatized in the same way.These reactions are represented below. After derivatization, urinesamples and calibration standards were evaporated to dryness undernitrogen and redissolved in 100 μl methanol for LC-MS analysis. Thereactions that produce the TSH derivatives are shown below.

G. HPLC-MS

LC-MS analysis was performed on a Finnigan LCQ™ DECA ion trap massspectrometer with Surveyor HPLC system (ThermoFinnigan, San Jose,Calif., USA) controlled by the Xcalibur software. Liquid chromatographywas carried out on a reverse phase Luna C18(2) column (150×2.0 mm, 3 μm;Phenomenex, Torrance, Calif., USA). The mobile phase consisted ofmethanol as solvent A and water with 0.1% (v/v) formic acid as solventB. The LC flow rate of 200 μl/min was used for both ESI and APCI modes.Sensitivity was such that only 5 μl of each 100-μl sample was injectedby autosampler for analysis. The entire chromatography effluent waspassed into the mass spectrometer interface for subsequent detection.

For the analysis of CE-TSH and d-CE-TSH, a linear gradient of A/Bchanging from 60:40 to 75:25 in 15 min was employed. After changing backfrom 75:25 to 60:40 in 2 min, the mobile phase composition A/B stayed at60:40 for 8 min before the next injection. The ESI positive ion mode wasused as follows: ion source voltage, 5 kV; heated capillary temperature,250° C.; capillary voltage, 15 V; sheath gas flow rate, 70 units;auxiliary gas flow rate, 15 units; tube lens offset, 50 V. MS full scanmode was employed for characterizing mass spectra of CE-TSH and d-CE-TSH(FIGS. 2A-2D). These mass spectra were used to identify appropriatemasses for detection of the compounds. MS selected ion monitoring (SIM)mode was used for the quantitative analysis. The protonated analyte ions[MH⁺], m/z 455 and m/z 459, were monitored for CE-TSH and d-CE-TSH,respectively. The less abundant natriated analyte ions [MNa⁺], about15-20% of [MH⁺], were used as the second ion pairs for confirming theanalyte identification. Similar results were obtained for a simpleisocratic elution solvent comprising 60% methanol/40% water with 0.1%(v/v) formic acid.

For the purpose of comparison, the LC-MS performance of CE and d-CEseparation/detection without TSH derivatization were also examined. Alinear gradient of A/B changing from 40:60 to 60:40 in 10 min wasemployed, and then held at 60:40 for an additional 10 min. Afterchanging back from 60:40 to 40:60 in 2 min, the mobile phase compositionA/B stayed at 40:60 for 8 min before the next injection. The APCIpositive ion mode was used as follows: ion source current, 10 μA;vaporizer temperature, 450° C.; heated capillary temperature, 175° C.;capillary voltage, 15 V; sheath gas flow rate, 80 units; tube lensoffset, 30 V. MS full scan mode was employed for characterizing the massspectra of CE and d-CE (FIGS. 4A-D). MS SIM mode was used for theanalysis of calibration standards without TSH derivatization. Theprotonated analyte ions [MH⁺], m/z 287 and m/z 291, were monitored forCE and d-CE, respectively.

H. Quantitation of CE

CE-TSH/d-CE-TSH area ratios were determined for the SIM chromatographicpeaks using the Xcalibur software. Calibration curves were constructedby plotting CE-TSH/d-CE-TSH peak area ratios obtained from calibrationstandards versus CE concentrations and fitting these data using linearregression. CE concentrations in urine samples were then interpolatedusing this linear function.

I. Absolute Recovery of CE After Hydrolysis and Extraction Procedure

To one set of six 10-ml aliquots of the pooled postmenopausal urine, 50μl of the d-CE working internal standard solution (40 ng d-CE) wasadded, followed by the hydrolysis and extraction procedure describedabove. A second set of six 10-ml aliquots of the pooled postmenopausalurine was treated identically, except that the d-CE was added after thehydrolysis and extraction procedure instead of at the beginning. Bothsets of samples were then derivatized and analyzed in consecutive LC-MSanalyses. The absolute recovery of CE after the hydrolysis andextraction procedure was calculated by dividing the CE-TSH/d-CE-TSH peakarea ratio from a sample of the second set with that from a comparablesample of the first set, and then calculating the mean of the sixvalues.

J. Accuracy and Precision of the Urinary CE Analysis

To assess accuracy and intra batch precision of the method, 50 μl of thed-CE working internal standard solution (40 ng d-CE) was added to eachof eighteen 10-ml aliquots of the pooled postmenopausal urine. Then,identical known amounts of CE (0, 8 or 30 ng, respectively) were addedto each of six urine aliquots. All the urine samples were hydrolyzed,extracted, derivatized, and analyzed as described above. The endogenousCE concentration for the pooled postmenopausal urine was determined asthe mean of the measured values from the six blank samples. Thisbaseline CE concentration was then subtracted from the values determinedfor CE spiked urine samples to assess method accuracy and intra batchprecision. In addition, duplicate aliquots of the pooled urines fromboth postmenopausal and premenopausal midluteal phase women werehydrolyzed, extracted, derivatized, and analyzed in four differentbatches to further assess the inter batch precision of the urinary CEanalysis.

K. ESI and APCI Mass Spectra

The ESI mass spectra for CE-TSH and d-CE-TSH derivatives (morespecifically the TSH derivatives of non-deuterated and deuterated2-hydroxyE₁ and 4-hydroxyE₁) are presented in FIGS. 2A-D. These spectraare characterized by intense protonated analyte ions [MH⁺] at m/z 455and m/z 459 for CE-TSH and d-CE-TSH, respectively, and less abundantnatriated analyte ions [MNa⁺], about 15-20% of [MH⁺], at m/z 477 and m/z481 for CE-TSH and d-CE-TSH, respectively. Based on these data, theprotonated analyte ions [MH⁺] were monitored for quantitative analysisin SIM mode, and natriated analyte ions [MNa⁺] were used as the secondion pairs for confirming the analyte identification. Note that littlefragmentation is seen in these spectra, indicating that the TSHderivatives are stable under the ESI conditions.

Since the sensitivity of LC-MS analysis for CE and d-CE withoutderivatization is poor during ESI, the APCI mode was chosen for theiranalysis. The APCI mass spectra for CE and d-CE without derivatizationare shown in FIGS. 3A-D. Unlike in the ESI mass spectra for CE-TSH andd-CE-TSH, the spectra of [MH⁺-H₂O], [MH⁺-2H₂O] and various steroid ringfragments were also observed in addition to [MH⁺]. The protonatedanalyte ions [MH⁺], m/z 287 and m/z 291, were monitored for CE and d-CE,respectively, during SIM mode analysis.

L. Importance of TSH Derivatization in CE Analysis

The success of TSH derivatization in CE analysis, and its importance isshown in FIGS. 4A and B. Comparison of FIGS. 4A and 4B reveals how TSHderivatization improved the peak separation and shortened thechromatography time. Within 11 min, baseline separation of2-hydroxyE₁-TSH and 4-hydroxyE₁-TSH was achieved with a difference inretention times of more than 1 min, whereas underivatized 2-hydroxyE₁and 4-hydroxyE₁ did not begin eluting until 15 min after injection andwere still not fully separated, with a difference in retention times ofless than 0.6 min, on the same C₁₈ column. Second, TSH derivatizationimproved HPLC column retention of the analytes. Therefore, a highermobile phase methanol composition could be employed for chromatographyof CE-TSH and d-CE-TSH, which improved the efficiency of the ESI processand enhanced method sensitivity, compared with CE and d-CE withoutderivatization. Third, TSH derivatization of CE and d-CE resulted instable and intense protonated analyte ions [MH⁺] with no fragmentationduring their ionization process (see FIGS. 2A-D), which contributed tothe improved sensitivity. Finally, the sulfonhydrazone in CE-TSH andd-CE-TSH has greater proton affinity than the ketone in CE and d-CE.This greatly enhanced the method sensitivity for ESI positive ion mode.

M. Chromatographic SIM Profiles of CE-TSH and d-CE-TSH in Standards andPooled Human Urine

Even though sample preparation in the disclosed method is substantiallysimplified, compared with the published stable isotope dilution GC-MSmethod, it is adequate for quantitative analysis of endogenous CE inpostmenopausal urine. The HPLC-ESI-MS SIM chromatographic profiles for a0-ng working standard appropriately gave no signal for the undeuteratedCE-TSH (not shown). Analyses of a 1-ng working standard, and a blankpostmenopausal urine sample are shown, respectively, in FIGS. 5A and B.Using a simple methanol-water reverse phase HPLC linear gradient,2-hydroxyE₁-TSH and 4-hydroxyE₁-TSH were eluted from the C₁₈ column inabout 9.5 and 10.6 min, respectively, with symmetrical peak shapes.CE-TSH was readily detected and quantified, with no interference, evenat the low endogenous levels in postmenopausal urine (FIG. 5B).

N. Standard Curve and Limit of Quantitation

Standard curves for CE were linear over a 100-fold calibration range(0.5-64 ng CE/sample) with correlation coefficients for the linearregression curves typically 0.999 (FIGS. 6A-B). Replicate (n=6)injections of a 1-ng working standard, representing 50 pg on column,resulted in Relative Standard Deviations (R.S.D.) of SIM peak arearatios for 2-hydroxyE₁-TSH/[²H₄] 2-hydroxyE₁-TSH and4-hydroxyE₁-TSH/[²H₄] 4-hydroxyE₁-TSH of 1.0 and 1.6%, respectively. TheSignal to Noise (S/N) ratios obtained for the 1-ng working standard,representing 50 pg on column, were typically greater than 15 (FIG. 5A),which provides an adequate lower limit of quantitation for endogenous CEanalyses in urine from postmenopausal women.

O. Absolute Recovery of CE after Hydrolysis and Extraction Procedure

The absolute recovery of CE after the hydrolysis and extractionprocedure was determined by comparing SIM chromatographic peak arearatios of CE-TSH/d-CE-TSH in pooled urine from postmenopausal women thathad been spiked with d-CE before and after the hydrolysis and extractionprocedure. Mean absolute recoveries were 82.4±2.9% and 81.5±2.5%,respectively, for 2-hydroxyE₁ and 4-hydroxyE₁.

P. Accuracy and Precision of the Urinary CE Analysis

Accuracy, intra and inter batch precision data for the stable isotopedilution HPLC-ESI-MS SIM analysis of human urine samples are presentedin Tables 1 and 2 below. The analysis of six 10-ml aliquots of thepooled postmenopausal urine generated a mean concentration forendogenous 2-hydroxyE₁ and 4-hydroxyE₁ of 9.64 ng/10 ml and 1.40 ng/10ml, respectively (Table 1). Subtraction of these baseline values fromthe mean concentrations of six identical postmenopausal urine aliquotsto which 8 ng or 30 ng of CE had been added led to the estimates ofaccuracy, which was 98.76 and 97.06% for 2-hydroxyE₁ and 98.01 and98.99% for 4-hydroxyE₁, respectively (Table 1). The intra batchprecision, as estimated by the R.S.D. from 6 replicate analyses at eachlevel, ranged from 1.64 to 3.25% for 2-hydroxyE₁ and 1.05 to 4.73% for4-hydroxyE₁, respectively (Table 1).

TABLE 1 Accuracy and intra batch precision of urinary CE analyses,including hydrolysis, extraction, and derivatization steps^(a)Postmenopausal Postmenopausal Postmenopausal urine urine + 8 ng CEurine + 30 ng CE 4- 2- 4- 2- 4- 2-hydroxyE₁ hydroxyE₁ hydroxyE₁hydroxyE₁ hydroxyE₁ hydroxyE₁ Mean (n = 6) 9.64 1.40 17.54 9.24 38.7631.10 SD (n = 6) 0.31 0.07 0.29 0.18 1.15 0.33 Accuracy N/A N/A 98.7698.01 97.06 98.99 (%) ^(a)The mean is expressed in units of ng/10 mL ofurine.

The inter batch precision estimated by the R.S.D. for 4 independentbatch analyses of pooled postmenopausal and premenopausal midlutealurine samples were 2.36 and 2.37% for 2-hydroxyE₁ and 4.44 and 10.68%for 4-hydroxyE₁, respectively (Table 2).

TABLE 2 Inter-batch precision of urinary CE analyses, includinghydrolysis, extraction, and derivatization steps^(a) PostmenopausalPremenopausal urine mid-luteal urine 2-hydroxyE₁ 4-hydroxyE₁ 2-hydroxyE₁4-hydroxyE₁ Mean (n = 4) 9.78 1.34 32.46 3.95 SD (n = 4) 0.23 0.06 0.770.42 Precision (%) 2.36 4.44 2.37 10.68 ^(a)The mean is expressed inunits of ng/10 ml urine.Q. Application to Pre- and Postmenopausal Urine Samples

The usefulness of the disclosed method was also demonstrated in theanalyses of endogenous CE in the non-pooled urine samples from twopostmenopausal women and two premenopausal women during midfollicularand midluteal phases as described above. Duplicate 10-ml aliquots fromeach 24-h urine sample were hydrolyzed, extracted, derivatized, andanalyzed to determine CE concentration. When this information wascombined with the associated 24-h urine volume, it provided estimates of24-h urinary CE excretion (2CE=2-hydroxyE1; 4CE=4-hydroxyE₁) in each ofthe postmenopausal women (Post-M) and premenopausal women duringmidfollicular (Pre-MF) and midluteal phases (Pre-ML) (FIG. 7). Thesedata correspond with the results of other reported studies (see, forexample, Aldercruetz et al., J. Natl. Cancer Inst., 86: 1076, 1994).

The HPLC-ESI-MS method for measuring endogenous CE in human urinedescribed above simplifies sample preparation and increases thethroughput of analysis. The disclosed method provides a simple and rapidderivatization step that forms p-toluenesulfonhydrazone derivatives ofCE and d-CE. This derivatization step greatly enhances ESI-MSsensitivity as well as HPLC separability of the 2- and 4-hydroxyE₁.Standard curves were linear over a 100-fold calibration range (0.5-64 ngCE/sample) with correlation coefficients for the linear regressioncurves typically 0.999. The lower limit of quantitation for each CE is 1ng per 10-ml urine sample, with accuracy of 97-99% and overallprecision, including the necessary preparation and derivatization steps,of 1-3% for samples prepared concurrently and 2-11% for samples preparedin several batches. This method is useful for measuring the lowendogenous levels of 2- and 4-hydroxyE₁ in urine from postmenopausalwomen.

Example 2 Derivatization Agents and Methods

Examples of sulfonhydrazide compounds useful for forming ESI-MSdetectable derivatives of carbonyl-containing compounds have thestructure

where R is selected from the group consisting of alkyl, substitutedalkyl, aryl, and substituted aryl. Alkyl groups includes C1-C18 straightand branched chain alkyl groups (for example C1-C10 or C1-C5 groups).Substituted alkyl includes alkyl groups in which one or more hydrogensare substituted with halogen (F, Cl, Br, I), amino groups or hydroxylgroups. Aryl includes phenyl, napthyl, and anthranyl groups. Substitutedaryl includes phenyl, napthyl, and anthranyl groups where one or morehydrogens are substituted with C1-C5 alkyl, C1-C4 alkoxy, halogen,amino, nitro, hydroxyl, carbonyl, nitroso, cyano, and sulfonyl groups,and combinations thereof.

In some embodiments, the sulfonhydrazide compound has the structure

wherein R₁-R₅ are independently selected from the group consisting ofhydrogen, C₁-C₅ alkyl, C1-C4 alkoxy, halogen, amino, nitro, hydroxyl,carbonyl, nitroso, cyano, and sulfonyl, and combinations thereof. C1-C5alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, and neopentyl groups. C1-C4 alkoxy includes,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy,and tert-butoxy groups. One example of a sulfonhydrazide compound havingthis structure is p-toluenesulfonhydrazide, wherein R₃ is methyl and R₁,R₂, R₄, and R₅ are hydrogen. Another example is benzenesulfonylhydrazide where R₁-R₅ are all hydrogen. Both p-toluenesulfonhydrazideand benzenesulfony hydrazide are available from Aldrich (Milwaukee,Wis.), as are 2,4,6-triisopropylbensenesulfonyl hydrazide,2,4,6-trimethylbensenesulfonyl hydrazide, 4-methoxybenzenesulfonylhydrazide, and 4-amino-2-nitrobenzenesulfonyl hydrazide. Othersulfonhydrazides may be synthesized by reacting a sulfonyl halide (suchas sulfonyl chloride) compound with hydrazine, an amine (see,Streitwieser and Heathcock, “Introduction to Organic Chemistry,”Macmillan Publishing Co., Inc., 1976, pages 789-790). For examplemethanesulfonyl chloride may be reacted with hydrazine to formmethanesulfonyl hydrazide. Over 150 sulfonyl chloride compounds areavailable from Aldrich (Milwaukee, Wis.). Others may be synthesized fromsulfonic acid compounds by reaction with PCl₅.

Sulfonhydrazide compounds may be reacted with carbonyl-containingcompounds by combining the two compounds in a solvent that will dissolveboth but does not itself react with the sulfonhydrazide compound (forexample, acetone would be a bad choice). Methanol is a good solvent inwhich to react sulfonhydrazide compounds with carbonyl compounds, suchas ketosteroids, because it is a good solvent for both polar andnon-polar solutes and it will not react with the sulfonhydrazidecompound. Once combined the reaction mixture may be heated to acceleratethe reaction, for example, to between 30° C. and 65° C. (reflux) whenmethanol is the solvent. A general procedure for formingp-toluenesulfonhydrazone derivatives of carbonyl compounds in methanolmay be found in Banwell et al., J. Chem. Soc. Perkin Trans., 1: 945,1993. In this method, the carbonyl containing compound is combined withthe sulfonhydrazide compound in methanol that is initially warmed to 50°C. The reaction is then left to sit at room temperature for 12-24 h.Another example is found in Banks et al., J. Am. Chem. Soc.,115:2473-2477, 1993, where ethanol is substituted for methanol and thereaction mixture containing the sulfonhydrazide compound and thecarbonyl-containing compound is refluxed (78° C.) for about 4 hours. Thetime of reaction will depend upon the particular carbonyl-containingcompound and the temperature (such as between about 25° C. and 100° C.)of the reaction mixture, but may vary from 15 minutes to about 2 days,with 30 minutes to a few hours being typical.

For TSH derivatization of estrogens, a series of TSH concentrations,reaction times, temperature of reaction and addition of an acid wereinvestigated to determine appropriate reaction conditions forderivatization. Estrogen samples were incubated at 60° C. for 0.5, 1,1.5 or 2 hours in 200 μL of 0.5 mg TSH/mL 100% methanol, 1 mg TSH/mL100% methanol or 2 mg TSH/mL 100% methanol. Improved derivatization wasobtained using the 2 mg TSH/mL 100% methanol solution, which showed nosignificant difference between the incubation periods. Estrogen sampleswere also incubated with 2 mg TSH/mL solutions in methanol that alsocontained 0.05 M or 0.10 M acetic acid. These samples showed improvedintensity of the protonated molecular ions. Different temperatures (45,50, 55 and 60° C.) were tried while holding the TSH concentration andtime constant, and revealed that derivatization was more effective at55° C. and 60° C. The TSH-estrogen adducts were also observed to bequite stable. When incubation mixtures were left at room temperature forperiods of 24-72 hours, little degradation or decomposition wasobserved.

Example 3 Ketosteroids

As used herein, ketosteroids include steroids having at least onecarbonyl group. Groups of steroids that include numbers of ketosteroidsare androgens, corticoids, estrogens, sterols, vitamin D metabolites,phytosteroids, neurosteroids and bile acids (see, for example, Shimadaet al., J. Chromatogr. A, 935: 141-172, 2001, for a discussion of themany types of steroids and many particular examples of ketosteroids).The basic steroid molecular skeleton consists of four rings of carbonatoms, perhydro-1,2-cyclopentenophenanthrene. Many steroids fall withinsix broad groups according to the number of carbon atoms in the 4-ringskeleton and in side chains, namely, gonanes (C17), estranes (C18),androstanes (C19), pregnanes (C21), cholanes (C24), and cholestanes(C27). Any of these skeletons bearing a carbonyl group is also aketosteroid. Particular examples of ketosteroids include thecarbonyl-bearing testosterones, testosterone esters, androsterones,norandrosterones, noretiocholanolones, cortisols, cortisones,aldosterones, corticosterones, tetrahydrocortisones, etiocholanolonespregnenolones, protesterones, estrones, gestrinones, oxosterols, guaicolestrogens, and metabolites of these compounds. Ketosteroids may benaturally occurring or synthetic, making the methods of the disclosureapplicable to metabolic studies as well as for detecting abuse ofperformance enhancing steroids.

Example 4 HPLC

The principles of chromatography, such as liquid chromatography, forexample high-performance liquid chromatography and its more sensitivevariants, nano-LC and capillary HPLC, are described in depth in severalexcellent textbooks including Scott, Techniques and Practices ofChromatogrphy, Marcel Dekker 1995; Meyer, Practical High-performanceLiquid Chromatography, 2^(nd) Ed., Wiley, New York, 1994; McMaster,“HPLC: A Practical User's Guide, VCH Publishers, Inc., 1994; andKrustulovic and Brown, Reversed-Phase HPLC: Theory, Practice andBiomedical Applications, Wiley-Interscience, New York, 1982. Nano-LC isalso described in a review article by Guetens et al. (Guetens et al., J.Chromatogr. B, 739: 139-150, 2000). A discussion of coupled liquidchromatography and mass spectrometry is found in Niessen and van derGreef, Liquid Chromatography-Mass Spectrometry, Marcel Dekker, Inc.,1992.

Briefly, HPLC is a form of liquid chromatography, meaning the mobilephase is a liquid. The stationary phase used in HPLC is typically asolid, more typically a derivatized solid having groups that impart ahydrophilic or hydrophobic character to the solid. For example, silicagel is often used as the base solid and it is derivatized to alter itsnormally hydrophobic characteristics. Normal phase HPLC refers to usinga non-polar mobile phase and a polar stationary phase. Reverse phaseHPLC refers to a polar mobile phase and a non-polar stationary phase.Reverse phase HPLC is convenient because polar solvents such as water,methanol, and ethanol may be used and these solvents are easily andsafely handled and disposed. Furthermore, reverse phase conditionsimprove ESI efficiency.

Typical reverse phase mobile phase solvents include polar proticsolvents such as water, methanol, ethanol, and sometimes other alcoholsand polar aprotic solvents such as dimethylformamide and acetonitrile.Of these, methanol and water are particularly convenient to use,especially since they are miscible in all proportions with each other.When a single solvent system (either a single solvent or a mixture ofsolvents) is used the chromatography is termed isocratic. When thecomposition of the mobile phase solvent is changed during achromatographic run it is termed a gradient elution. For reverse phaseHPLC, a gradient begins with the more polar solvent mixture and thenprogressively is changed to a more non-polar solvent system. Forexample, a reverse phase gradient elution may begin with a 20:80methanol/water mixture and change to an 80:20 methanol/water mixtureduring the course of a chromatographic run. Other examples ofmethanol/water mixture gradients include beginning with a 25:75methanol/water mixture and changing to 75:25 methanol/water during thechromatographic run or beginning with a 40:60 methanol/water solvent andchanging to a 60:40 methanol/water mixture. Formic acid may also beadded to the solvent in an amount from 0.05% to 1%, such as from 0.05%to 0.2%, to assist in positive ion mode ESI of the HPLC effluent.

A convenient isocratic solvent system for separation ofp-toluenesulfonhydrazide derivatized catechol estrogens is a 60:40methanol/water mixture. Such a solvent system has an espsilon value(solvent strength parameter) of about 51. One of ordinary skill in theart of chromatography will recognize that other solvent systems ofsimilar epsilon value can be chosen to accomplish the separation. Forexample, a 50:50 acetonitrile/water mixture has an epsilon value of 50and may be chosen as an alternative solvent system. Similarly,equivalents for other methanol/water solvents that are better suited forparticular ketosteroid separations may be chosen on the basis of epsilonvalues. Mixtures of solvents may be mixed beforehand or be mixed duringa chromatographic run in varying proportions in what is called agradient elution. In general, appropriate solvent systems and gradientsfor reverse phase HPLC will have an epsilon value from 30 to 80, forexample, from 40 to 70, such as from 45 to 55. Examples of solvents thatmay be used to provide an epsilon values from 30 to 80 include water,methanol, ethanol, and acetonitrile.

As stated above, reverse phase HPLC is better suited to ESI conditionsthan normal phase HPLC. For reverse phase HPLC, the non-polar stationaryphase may be a C8 or a C18 derivatized column or an embeddedpolar/non-polar column such as an amine/C18 or C8 column. In theembedded column, polar groups close to the surface of the solidstationary phase support are interspersed with non-polar C8 or C18groups. Many types of non-polar columns for reverse phase HPLC areavailable, for example, from Alltech Associates, Inc. (Deerfield, Ill.).

In some instances it may be desirable to utilize a nano-LC (capillaryHPLC) technique to increase sensitivity of the disclosed methods. NanoLC is often combined with online mass spectrometry using micro- ornano-ion spray (variants of ESI). Nano-LC columns are available in avariety of sizes and lengths. For example, a typical column might havean inner diameter of 75 μm and a length of between 5 and 25 cm. Atypical nano-LC packing is C18, with a 5 μm particle size, making itespecially suitable for separations of ketosteroids according to thedisclosed methods. Nano-LC equipment is available, for example, from LCPackings (San Francisco, Calif.).

Example 5 Derivatization for ESI

It is also possible to use sulfonhydrazide compounds to increase theionization efficiency of carbonyl-containing compounds under positiveion mode ESI conditions and thereby lower the limit of detection of thecompound by ESI-MS. In this embodiment, a sulfonhydrazide compound isadded to a liquid sample containing a carbonyl-containing compound andallowed to react to form a sulfonhydrazone derivative of thecarbonyl-containing compound. The sample is then injected in liquid form(with or without further purification) into an ESI-MS device formeasurement (see for example, Fenn et al., “ElectrosprayIonization-Principles and Practice,” Mass Spectrom. Rev., 9: 37-70,1990).

Example 6 Mass Spectrometers

An API interface (such as an ESI interface) may be used to introduce aliquid sample into any type of mass spectrometer in API-MS. Examples ofthe types of mass spectrometers that may be used include sectorinstruments, quadrupole instruments, ion-cyclotron resonanceinstruments, time-of-flight instruments, and tandem mass spectrometers.A particularly useful type of tandem mass spectrometer for ESI ionizedsamples is an instrument having a collision cell, such as a low-energyor high-energy collision cell, placed between the mass selecting regionsof the spectrometer. Since ESI typically creates ions from moleculeswithout breaking them apart, it is advantageous to break the ions apartinto fragment ions in a collision cell. The fragmentation patterncreated in the collision cell is detected by a second mass selectivedevice and may be used for identification of the analyte.

Example 7 Comparison of Sulfonhydrazide Derivatization with OtherDerivatization Schemes

To demonstrate the exceptional signal enhancing ability ofsulfonhydrazide derivatization for API-MS and the advantageous HPLCproperties of p-toluenesulfonhydrazones derived from ketosteroids, othertypes of derivatives were prepared and tested. Methoxyamine andethoxyamine derivatization of the carbonyl group of catechol estrogensproduced no detectable signal in either ESI or Atmospheric PressureChemical Ionization (ACPI) experiments. Carboxymethoxylamine (CMA)formed the derivatized product and it was detectable in both ESI andACPI experiments. However, CMA derivatives were not stable during eitherESI or ACPI and decomposed, making quantification more difficult.Girard's Reagent P and T (quaternary ammonium hydrazine compounds with apermanent positive charge) derivatives showed improved signal for ESI,but they adversely affected the HPLC separation of catechol estrogens.6-Ethoxy-2-benzothiazolesulfonamide, N′-(2-thiazolyl)sulfanilamide,sulfisomididine, and sulfadiazine were also tried as derivatizationagents, but no signal was observed in either ESI or ACPI.Sulfonyhydrazide derivatization, by comparison, showed the bestenhancement of the ESI signal, and p-toluenesulfonylhydrazidederivatization in particular was most effective for improving thechromatographic behavior of the ketosteroid catechol estrogens.

Example 8 Combining Sulfonhydrazide Derivatization and SulfonylDerivatization

In this example, carbonyl protection via derivatization with asulfahydrazide in combination with hydroxyl protection viaderivatization with a sulfonyl halide allows the detection of bothcarbonyl and hydroxyl containing steroids, such as estrogens andandrogens, and their metabolites. For example, only some endogenousestrogens and their metabolites possess a carbonyl functional group. Incontrast, all endogenous estrogens and their metabolites have a phenolicor catechol A-ring. Addition to the hydroxyl of a highly ionizablemoiety such as a dansyl group can be performed by reaction with asulfonyl halide. Such a step will add the highly ionizable moiety to allendogenous estrogens, because all these estrogens have a hydroxyl groupthat can be derivatized by the reaction. In contrast, not all endogenousestrogens contain a carbonyl group, and in the absence of the carbonylgroup they are not capable of being derivatized by the sulfonhydrazide.Derivatization with the sulfonhydrazide alone is therefore not able toderivatize all endogenous estrogens for the purpose of enhancing theirAPI (such as ESI) efficiency and HPLC-ESI-MS method sensitivity atpositive ion mode.

Although it is advantageous to perform sulfonyl derivatization of thehydroxyl groups of the endogenous steroids (such as estrogens orandrogens), it is difficult to use such an approach with steroids thatcontain the carbonyl. The carbonyl group in D-ring ketolic steroidmetabolites (such as estrogen) is alkali labile, and is readilydestroyed or interconverted in an alkaline environment during thereaction with the sulfonyl halide. In this example, the inventorsdisclose protecting the carbonyl group in D-ring ketolic estrogenmetabolites by reacting with a sulfonhydrazide to form a chemicallystable hydrazone before carrying out the sulfonyl derivatization. Thismethod allows the derivatization of the hydroxyl group while avoidingcarbonyl degradation under the alkali conditions of the sulfonylderivatization. This combined derivatization approach allows both thehydroxyl and carbonyl groups to be derivatized without altering thecarbonyl groups of the steroids.

Examples of the sulfonhyrazide compound are listed in Example 2, and ina specific example is TSH.

The sulfonyl derivatizing agents (such as sulfonyl halides) useful forforming API-MS (such as ESI-MS) detectable derivatives ofhydroxyl-containing compounds in this example have the structure

wherein X is Cl, Br, I, or any good leaving group, and R is any highlyionizable group that allow for the formation of ions under either thepositive ion mode or the negative mode of ESI-MS, such as alkyl;substituted alkyl, aryl, and substituted aryl. In particular embodimentsthe alkyl is a lower alkyl (a C1-C10 alkyl, such as a C1-C5 alkyl).

In some embodiments, the sulfonyl derivatizing agent is a sulfonylhalide, such as a having the structure

wherein X is Cl, Br, I, or any good leaving group, and R1-R8 areindependently selected from the group consisting of hydrogen, C1-C5alkyl, C1-C4 alkoxy, halogen, amino, nitro, hydroxyl, carbonyl, nitroso,cyano, and sulfonyl, and combinations thereof. In particular examples,R1-R6 are all hydrogen, and R7 and R8 are lower alkyl.

One example of a sulfonyl halide compound having this structure is adansyl halide such as dansyl chloride which has the following chemicalstructure.

A. Chemicals and Reagents

Sixteen estrogens and estrogen metabolites (EM), as shown below, wereobtained from Steraloids, Inc. (Newport, R.I., USA). Deuterium-labeledestrogens and estrogen metabolites (d-EM) were purchased from C/D/NIsotopes, Inc. (Pointe-Claire, Quebec, Canada). All EM and d-EManalytical standards have chemical and isotopic purity ≧98%,respectively, as reported by the manufacturers, and were used withoutfurther purifications.

p-Toluenesulfonhydrazide (TSH), dansyl chloride, and acetone (HPLCgrade) were purchased from Aldrich Chemical Co. (Milwaukee, Wis., USA).Methanol (HPLC grade) and formic acid (reagent grade) were obtained fromEM Science (Gibbstown, N.J., USA), and water (HPLC grade) was obtainedfrom Mallinckrodt Baker, Inc. (Paris, Ky., USA). Sodium bicarbonate(reagent grade) and sodium hydroxide (reagent grade) were purchased fromJ. T. Baker (Phillipsburg, N.J., USA).

B. Preparation of Stock and Working Standard Solutions

Stock solutions of EM and d-EM were prepared at 80 μg ml⁻¹ by additionof 2 mg individual estrogen powder to a volumetric flask and diluting to25 ml with 100% methanol. These solutions were stored at −20° C. untilneeded to prepare working standard solutions. During each day ofanalysis, working standards of EM and d-EM were freshly prepared byserial dilutions of stock solutions with 100% methanol. In this example,d-EM working standard solution was prepared at 800 ng ml⁻¹, while EMworking standard solutions were prepared at 800 and 50 ng ml⁻¹.

C. Derivatization Procedure

The methanolic solutions containing 8 ng of EM or d-EM was evaporated todryness under nitrogen gas (Reacti-Vap III™, Pierce, Rockford, Ill.,USA) and reacted with 400 μg p-toluenesulfonhydrazide in 200 μl methanoland heating at 60° C. (Reacti-Therm III™ Heating Module, Pierce,Rockford, Ill., USA) for 30 min (Xu et al. 2002, J Chromatogr B) toprotect the carbonyl group. After TSH derivatization, estrogen sampleswere evaporated to dryness under nitrogen and redissolved in 70-μl ofdansyl chloride solution (1 mg ml⁻¹ in acetone) and 30-μl of 50 mMsodium bicarbonate buffer (pH=10.5) and heated at 50° C. for 5 min(Yamada et al. 2000, Biomed Chromatogr).

For the purpose of comparison, the same EM or d-EM were derivatized withdansyl chloride procedure alone without prior protection of carbonylgroup by TSH.

In both approaches, the reaction mixtures were directly injected forHPLC-ESI-MS analysis.

D. HPLC-ESI-MS

HPLC-ESI-MS analysis was performed on a Finnigan LCQ™ DECA ion trap massspectrometer with Surveyor HPLC system (ThermoFinnigan, San Jose,Calif., USA) controlled by the Xcalibur™ software. Liquid chromatographywas carried out on a reverse phase Luna C₁₈ column (150×2.0 mm, 3 μm;Phenomenex, Torrance, Calif., USA). The mobile phase consisted ofmethanol and water (85:15) with 0.1% (v/v) formic acid at the flow rateof 200 μl/min was used. Sensitivity was such that only 5 μl of each100-μl sample was injected by autosampler for analysis. The entirechromatography effluent was passed into the mass spectrometer ESIinterface for subsequent detection.

The ESI positive ion mode was used as follows: ion source voltage, 5 kV;heated capillary temperature, 250° C.; capillary voltage, 15 V; sheathgas flow rate, 70 units; auxiliary gas flow rate, 15 units; tube lensoffset, 50 V. MS full scan mode was employed for characterizing massspectra of both derivatization approaches.

E. Combining TSH Derivatization and Dansyl Halide Derivatization forImproved Signal Detection

With combined sulfonhydrazide derivatization of the carbonyl andsulfonyl derivatization of the hydroxyl (with TSH and dansyl chloriderespectively in this particular example), all estrogens and estrogenmetabolites show intense protonated molecule [MH⁺] and less abundantnatriated molecule [MNa⁺] during ESI positive ion mode. In contrast,some D-ring ketolic estrogen metabolites, such as 16α-hydroxyestrone,16-ketoestradiol, 2-hydroxyestrone, and 4-hydroxyestrone, show ratherpoor response and hydrogen loss when derivatized with the dansylchloride procedure alone without prior carbonyl protection (FIG. 8),which is believed to be due to their alkali labile nature. The extensivehydrogen loss found in deuterium-labelled D-ring ketolic estrogenmetabolites with dansyl chloride derivatization alone is especiallyproblematic since ion clusters from their protonated or natriatedmolecules also cover those of target analytes (FIGS. 8 and 9), whichmakes accurate quantitative measurement difficult.

Unlike the dansyl chloride derivatization alone, the combined TSHcarbonyl protection and dansyl chloride derivatization is a methodsuitable for the quantitative measurement of all endogenous steroids(such as estrogens and estrogen metabolites) by HPLC-ESI-MS.

Example 9 General Approach to Combining Carbonyl and HydroxylDerivatization of Steroids

In some more general embodiments, protecting the carbonyl group of thesteroids allows for the derivatization of the hydroxyl group withoutsignificant steroid degradation. This two-step process of carbonylderivatization followed by hydroxyl derivatization provides for betterHPLC separation of steroids, and allows for better signal detection inAPI-MS (such as ESI-MS) when at least one of the derivatization groupscontains a highly ionizable moiety that enables ionization under eitherpositive ion or negative ion mode of electrospray ionization.

Examples of carbonyl protecting reagents that could be used in thistwo-step process include but are not limited to compounds that form anoxime derivative, silyl derivative, ketal/acetal, hydrazone, andSchiff's base derivative. Specific examples include methoxyamine,ethoxyamine, carboxymethoxylamine, Girard's Reagent T, Giard's ReagentP, 6-ethoxy-2-benzothiazolesulfonamide, cystein,N′-(2-Thiazolyl)sulfanilamide, sulfisomidine, sulfadiazine, andp-toluenesulfohydrazide (TSH).

Examples of hydroxyl protecting reagent that could be used in thistwo-step process includes but not limited to compounds that form silylderivative, acyl derivative, benzoyl derivative, alkyl derivative,dansyl derivative, nitrobenzofuran derivative. Specific examples includenitrobenzopentaflurobenzoyl hydroxylamine, hydroxylamine, dabsylchloride, and dansyl chloride, 1-fluoro-2,4-dinitrobenzene, and4-fluoro-3nitrobenzofurazan.

In a particular specific example of this two step process, the carbonylgroup in D-ring ketolic steroid metabolites (such as estrogenmetabolites) is protected by forming a hydrazone, such as a chemicallystable p-toluenesulfonhydrazone, before carrying out a dansyl halidederivatization (for example with dansyl chloride) under alkalineconditions.

For carbonyl, the most useful protective groups are the acyclic andcyclic acetals or ketals, and the acyclic and cyclic thioacetals orketals. The carbonyl group can form a number of very stable derivativessuch as cyanohydrins, hydrazones, imines, oximes, and semicarbazones,which is suitable for derivatization.

It should be recognized that the illustrated embodiments are onlyparticular examples of the inventions and should not be taken as alimitation on the scope of the inventions. Rather, the inventionsinclude all that comes within the scope and spirit of the followingclaims.

1. A method for detecting ketosteroids, comprising: reacting a samplewith a sulfonhydrazide to form a sulfonhydrazone of a ketosteroid in thesample; reacting the sulfonhydrazone with a sulfonyl halide; andanalyzing the reacted sample by mass spectrometry to detect theketosteroid by detecting the sulfonyl halide derivative of thesulfonhydrazone of the ketosteroid, wherein detection of the sulfonylhalide derivative of the sulfonhydrazone indicates presence of theketosteroid.
 2. The method of claim 1, wherein analyzing the sample bymass spectrometry comprises atmospheric pressure ionization.
 3. Themethod of claim 2, wherein atmospheric pressure ionization comprisespositive ion mode electrospray ionization.
 4. The method of claim 1further comprising separating the ketosteroid from other components inthe sample by liquid chromatography.
 5. The method of claim 4, whereinthe liquid chromatography is high performance liquid chromatography(HPLC).
 6. The method of claim 4, wherein the ketosteroid is reactedwith the sulfonhydrazide prior to separating the ketosteroid by liquidchromatography.
 7. The method of claim 5, wherein separating theketosteroid from other components in the sample by HPLC comprisesreverse phase HPLC using a non-polar stationary phase.
 8. The method ofclaim 7 wherein reverse phase HPLC is performed using a methanol/watersolvent.
 9. The method of claim 7, wherein the non-polar stationaryphase is a C18 stationary phase.
 10. The method of claim 8, wherein HPLCis performed with gradient elution from 20:80 methanol/water to 80:20methanol/water is used.
 11. The method of claim 10, wherein gradientelution is performed from 40:60 methanol water to 60:40 methanol wateris used.
 12. The method of claim 1 further comprising extracting theketosteroid from the sample prior to reacting the sample with thesulfonhydrazide to provide a concentrated sample for analysis.
 13. Themethod of claim 1, wherein the ketosteroid is an estrogen.
 14. Themethod of claim 13, wherein the ketosteroid is a catechol estrogen. 15.The method of claim 1, wherein the sulfonhydrazide isp-toluenesulfonylhydrazide.
 16. The method of claim 1, wherein thesulfonyl halide comprises

wherein X is Cl, Br, or I, and R is alkyl, substituted alkyl, aryl, orsubstituted aryl.
 17. The method of claim 16, wherein R comprises loweralkyl.